Vascular endothelial growth factor (VEGF) is a major angiogenic factor in human malignancies, including breast cancer. VEGF-D is a recent member of the VEGF family, which stimulates vascular and lymphatic endothelial cell growth in experimental models but its function in the mammary gland is unknown. The goals of our project are the following:1) Characterize the functions of VEGF in mammary gland development and carcinogenesis, using transgenic mouse models with either overexpression or conditional knockout of VEGF in mammary epithelial cells; 2)Understand the mechanism by which VEGF acts as a survival factor for breast carcinoma cells; 3) Define the role of VEGF-D in mammary gland biology. 1.Transgenic expression of VEGF in mammary epithelial cells under the control of the mouse mammary tumor virus (MMTV) did not have morphological or functional impact on mammary gland development.Induction of mammary carcinomas was accomplished through breeding with MMTV-Middle T transgenic mice, producting VEGF/MT mice. Accelerated tumor development was observed in the VEGF/MT mice and the early tumors displayed striking vasodilatation. The established mammary carcinomas of the VEGF overexpressing mice showed increased stromal formation and vascular density, as well as decreased number of apoptotic cells and increased proliferative activity. There was also hemorrhage into tumor cavities which were lined with endothelial-like cells. Furthermore, the signaling VEGF receptor, Flk-1, and its co-receptor, neuropilin-1 (NRP-1), were expressed on the tumor cells and were upregulated in the VEGF/MT tumors. This is suggestive of an autocrine function of VEGF. VEGF overexpression was also associated with a significant increase in the number of lung metastases. Angiogenesis microarray data showed upregulation of all but one of the genes examined. There was a surprisingly strong correlation between the microarray data and genes reported to be upregulated VEGF in both in vivo and in vitro studies. Collectively the data from the transgenic studies indicate that VEGF can contribute in different ways to increased tumor growth and metastatic development, including vasodilatation, vascular permeability, increased vascularity, increased tumor cell proliferation, inhibition of apoptosis, and stimulation of multiple angiogenesis-associated genes. Inactivation of the VEGF gene in mammary epithelial cells in virgin mice had dramatic effects of development with almost complete cessation of milk production. When VEGF was deleted in late pregnancy, the areas of the mammary gland underwent unscheduled involution with apoptosis of the epithelial cells. This finding suggests that VEGF is not only a survival factor for tumor cells but also for normal mammary epithelial cells. Mammary carcinogenesis studies are still ongoing and show greatly delayed tumor induction in mice with deletion of VEGF at puperty. 2. Reported in vitro studies have suggested autocrine survival function of VEGF through binding to NRP-1. However, NRP-1 is not a signaling receptor and uses KDR/Flk-1 as a signaling co-receptor in endothelial cells, and plexin-A1 or L1-CAM in neuronal cells. We set out to examine distribution of NRP-1 and its known co-receptors in human breast carcinomas. We found overlapping expression of these receptors in serial histological sections of infiltrating ductal carcinomas. Confocal microscopic examination revealed co-localization of NRP-1 and plexin A-1 in the human breast cancer samples. Similar to the findings in the VEGF transgenic mouse model, we observed induction of NRP-1 in human breast carcinoma cells following VEGF transfection. Present focus is on further characterization of the receptors and signal transduction pathways, which VEGF utilizes to carry out its autocrine functions in cancer cells. We discovered a novel splice variant of human NRP-1, which encodes a soluble form of the protein (sNRP-1). Recent report on another splice variant of sNRP-1 demonstrated that it had anti-angiogenic effect on tumor xenografts. We will examine if our sNRP-1 suppresses paracrine and autocrine functions of VEGF. In parallel with in vitro studies to examine if sNRP-1 interferes with VEGF binding to the full-length NRP-1, in vivo studies will be carried out using transgenic mice expressing sNRP-1 in skeletal muscle, which results in high circulating levels of the protein. Founders of these mice have already been generated. 3. In human breast tissues, VEGF-D displayed different expression patterns, compared to that observed previously for VEGF. This entails higher VEGF-D mRNA levels in normal than malignant breast tissues and strong VEGF-D immunopositivity in blood vessels and normal ducts. Co-localization with alpha smooth muscle actin (SMA)established that VEGF-D is present in vascular smooth muscle cells and ductal myoepithelial cells. Corresponding with the Northern data of no or low VEGF-D mRNA expression in the invasive breast carcinomas, immunohistochemistry failed to detect VEGF-D in many of the breast cancer cases, while others showed weak VEGF-D positivity in cancer cells and scattered stromal fibroblasts. VEGF-D was co-localized with SMA in the stromal fibroblasts and well differentiated carcinomas with glandular growth pattern. In view of the smooth muscle cell localization of VEGF-D, we postulated that it acts as a growth factor for smooth muscle cells. Low passage aortic and bronchial smooth muscle cells expressed the signaling KDR/Flk-1 VEGF receptor and were growth stimulated by recombinant human VEGF-D to a similar degree as was observed for endothelial cells. Growth stimulation was associated with increased phosphorylation of KDR/Flk-1 and activation of the mitogen activated protein(MAP) kinase pathway. The role of VEGF-D in myoepithelial biology remains to be determined. An interesting finding of myoepithelial differentation of VEGF-D transfected human breast carcinoma cell lines suggests that it may be involved in differentiation of epithelial to myoepithelial cells.